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J. Agric. Food Chem. 2010, 58, 8560–8565 DOI:10.1021/jf1012456
Tremetone and Structurally Related Compounds in White Snakeroot (Ageratina altissima): A Plant Associated with Trembles and Milk Sickness STEPHEN T. LEE,*,† T. ZANE DAVIS,† DALE R. GARDNER,† STEVEN M. COLEGATE,† DANIEL COOK,† BENEDICT T. GREEN,† KIMBERLY A. MEYERHOLTZ,‡ CHRISTINA R. WILSON,‡ BRYAN L. STEGELMEIER,† AND TIM J. EVANS§ †
Poisonous Plant Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1150 East 1400 North, Logan, Utah 84341, ‡Indiana Animal Disease Diagnostic Laboratory, Purdue University, 406 South University, West Lafayette, Indiana 47907, and §Veterinary Medical Diagnostic Laboratory, University of Missouri;Columbia, 1600 East Rollins Street, Columbia, Missouri 65211
Ingestion of white snakeroot (Ageratina altissima) can cause trembles in livestock and milk sickness in humans. The toxicity has been associated with tremetol, a relatively crude, multicomponent lipophilic extract of the plant. In this study, 11 different compounds were isolated from white snakeroot-derived lipophilic extracts from 18 collections. Six of the isolated compounds have not been previously reported to be found in white snakeroot. High-performance liquid chromatography (HPLC) analysis indicated that there are three different chemotypes of white snakeroot from the plant samples analyzed. Elucidation of these chemotypes may explain the sporadic and unpredictable toxicity of white snakeroot to livestock and humans. KEYWORDS: White snakeroot; tremetol; Eupatorium rugosum; Eupatorium urticaefolium; Ageratina altissima; tremetone; dehydrotremetone
INTRODUCTION
White snakeroot (Ageratina altissima (L.) King & H. Rob. var. altissima, a member of the Asteraceae, previously named Eupatorium rugosum Houtt and Eupatorium urticaefolium Reichard), is commonly found throughout the eastern half of North America. Ingestion by livestock can sporadically and unpredictably cause a disease called trembles characterized by weight loss, listlessness, reluctance to move, muscular tremors when forced to stand and move, constipation, and apparent joint stiffness (1-3). Terminal collapse and coma can often occur in livestock several days to weeks after clinical signs appear (1, 2). Consumption of milk from cows that have grazed white snakeroot can cause milk sickness, a sometimes fatal disease in humans, even if lactating cows show few signs of sickness. Historically, milk sickness was also called sick stomach or the slows because it was characterized by listlessness, leg pains and cramps, loss of appetite, vomiting, constipation, a white coating on the tongue, and an acetone odor to the breath (3). Milk sickness caused many deaths among Midwestern settlers during the 1800s, ultimately forcing entire settlements to be abandoned (1, 2). In 1917, after nearly 100 years of investigations, white snakeroot was determined to be the cause of trembles and milk sickness (1). In the late 1920s, Couch (2) concluded that the toxin in white snakeroot was a compound which he named tremetol. However, tremetol was later determined to be a complex mixture including at least six components referred to as terpene I, sterol I, sterol II, tremetone (1), hydroxytremetone (2), and dehydrotremetone (3) *Corresponding author [phone (435) 752-2941; fax (435) 753-5681; e-mail
[email protected]].
pubs.acs.org/JAFC
Published on Web 07/20/2010
(Figure 1) (4). Tremetone (1), hydroxytremetone (2), and dehydrotremetone (3) were toxic in a goldfish bioassay and, therefore, it was concluded that these compounds were likely responsible for causing trembles and milk sickness (4). However, synthetic tremetone (1) was not toxic to white leghorn cockerels or sheep (5). More recently, Beier and co-workers (6, 7) reported that, following microsomal activation, tremetone (1), but not dehydrotremetone (2), was toxic in vitro to murine melanoma (B16F1) cells and five other mammalian cell lines. Thus, the relative toxicity of the white snakerootderived benzofuran compounds and sterols to livestock and humans remains elusive. The toxicity of white snakeroot has been difficult to study because some populations of plants appear to be nontoxic (3,8). Generalizations have been made that fresh plant from 1 to 20% of an animal’s body weight must be consumed to cause death (9,10). However, this has not been correlated to physiochemical concentrations. The objective of this study was to characterize the chemical profiles of white snakeroot from different populations. HPLC methods have allowed the isolation, identification, and quantification of 11 different compounds from plants in 18 geographically different white snakeroot populations. This information may provide insights into the sporadic and unpredictable nature of the intoxication related to white snakeroot. MATERIALS AND METHODS Plant Material. White snakeroot was collected from 18 different locations (Table 1). Composite collections of the above ground parts of at least 10 plants were collected randomly at each site except for the collection at Karst Trailhead, which was a collection of 6 plants. The plants collected
© 2010 American Chemical Society
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Figure 1. Chemical structures of tremetone (1), 6-hydroxytremetone (2), dehydrotremetone (3), dehydrotremetone (3), 2-senecioyl-4-acetylphenol (4), 2-senecioyl-4-(1-methoxyethyl)phenol (5), 6-acetyl-2,2-dimethylchroman-4-one (6), 6-acetyl-7-methoxy-2,2-dimethylchromene (7), 6-acetyl-8-methoxy-2,2dimethylchromene (8), 6-acetyl-5-hydroxy-8-methoxy-2,2-dimethylchromene (9), 6,7-dimethoxy-2,2-dimethylchromene (10), and 6-(1-hydroxyethyl)-7methoxy-2,2-dimethylchromene (11). were in early to full flower. White snakeroot plants were identified by Dr. Stanley L. Welsh, curator at the Stanley L. Welsh Herbarium at Brigham Young University, Provo, UT, and Dr. David S. Seigler, Professor, Department of Plant Biology, University of Illinois, Champaign, IL. The current taxonomic classification for this plant is given as Ageratina altissima (L.) King & H. Rob. var. altissima as per the USDA, National Resources Conservation Service, Plant Database. Voucher specimens for the white snakeroot were deposited in the Poisonous Plant Research Laboratory Herbarium, Logan, UT, and accession numbers are listed in Table 1. The plant collections from the Vermilion Research Observatory (VRO), Trelease Woods, Brownfield Woods, Allerton Park, Interstate, and Salt Fork sites in Illinois and those from the Davies County site in Missouri were air-dried at ambient temperature. The plant collections from the Rutan Woods, Stidham Woods, Hart Woods, and Tonica sites in Illinois and those from the Veterinary Medical Diagnostic Laboratory at the University of Missouri (VMDL), Karst Trailhead, Pierpont Meadows Road, Evans Place, Shooting Star Trailhead sites in Missouri, the Wabash River in Indiana, and the Cincinnati Zoo in Ohio were freeze-dried. The dried plant material was ground using a Cyclotec 1093 sample mill (Tecator, Hoganas, Sweden) to pass through a 1 mm screen. Extraction and Isolation of Compounds. Method A: ExtractionSilica Gel Column Chromatography-Preparative HPLC. Compounds 1 and 3 were previously isolated from Isocoma pluriflora (rayless goldenrod) (11). Using the same method, compounds 2 and 6 were isolated from white snakeroot collected at the Vermilion Research Observatory site and at the Trelease Woods site, respectively. For example, dry, ground,
white snakeroot (1.25 kg) was extracted for 112 h by Soxhlet extraction with hexane (9 L). The extract was rotary-evaporated to a viscous dark green residue. An aliquot of the residue were redissolved in CHCl3 and was adsorbed on approximately 75 g of silica (70-230 mesh, 60 A˚; SigmaAldrich, St. Louis, MO), and the solvent was allowed to evaporate. A 30 cm 8 cm i.d. silica (70-230 mesh, 60 A˚) column was prepared by slurry packing silica (650 g) in hexane/ethyl acetate (90:10) (1.5 L). The sample, adsorbed on silica, was then added to the head of the silica column. Sand (white quartz -50 þ 70 mesh; Sigma, St. Louis, MO) (2 cm) was added on top of the sample to protect the column bed. Mobile phase 1 (hexane/ethyl acetate 90:10, 1.5 L) was added to the head of the column until a first intense yellow band completely eluted. After elution of the yellow band, mobile phase 2 (hexane/ethyl acetate, 70:30, 2.5 L) was added to the column and the eluent collected. Mobile phase 2 eluent was rotaryevaporated to a dark green viscous residue, which was re-extracted with MeOH/H2O (70:30). This aqueous methanol extract was repeatedly filtered through a 30 mm nylon 0.45 μm syringe filter (National Scientific Co., Rockwood, TN) until clear and injected (2-5 mL, depending on the sample concentration) onto a Waters Prep LC2000 Preparative Chromatography System equipped with a UV-vis detector (Millipore Co., Milford, MA) monitoring λ 280 nm. The HPLC column (two 40 100 mm Bondapak C18 PrepPak cartridges (15-20 μm, 125 A˚; Millipore Co.) connected in series) was protected with a guard column (40 10 mm) of the same packing material. The mobile phase was 20 mM ammonium acetate/methanol (30:70, v/v) at a flow rate of 75 mL/min. The major peaks were collected separately from multiple injections. The combined
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Table 1. White Snakeroot Collection Sites, Dates, and Herbarium Accession Numbers collection site Illinois sites Stidham Woods Hart Woods Brownfield Woods Rutan Woods Salt Fork Interstate VRO Tonica Allerton Park Trelease Woods Missouri sites Davies County Shooting Star Trail Karst Trailhead VMDL Evans Place Pierpont Meadows Indiana site Wabash River Ohio site Cincinnati Zoo
county
GPS coordinates
collection date (month/day/year)
accession no.
Champaign Champaign Champaign Vermilion Vermilion Vermilion Vermilion LaSalle Piatt Champaign
40° 12.6670 40° 13.7320 40° 08.8740 40° 04.4060 40° 05.5360 40° 06.4830 40° 03.5870 41° 12.0110 40° 00.3660 40° 08.1110
N/88° 21.9700 W N/88° 21.3460 W N/88° 09.7860 W N/87° 54.3700 W N/87° 49.6830 W N/87° 40.8660 W N/87° 33.8860 W N/89° 01.2360 W N/88° 39.1830 W N/88° 08.3900 W
09/01/2009 09/28/2009 09/16/2009 09/01/2009 09/16/2009 09/17/2009 09/15/2009 08/15/2008 09/16/2009 09/16/2009
3546 3519 3558 3545 3555 3562 3550 3614 3560 3557
Davies Boone Boone Boone Boone Boone
39° 58.3000 38° 52.1670 38° 51.9840 38° 56.4100 38° 51.2280 38° 51.2860
N/93° 59.0160 N/92° 17.5270 N/92° 18.1950 N/92° 18.9330 N/92° 19.6110 N/92° 19.7010
W W W W W W
09/14/2009 09/04/2008 10/18/2007 10/01/2009 10/01/2009 09/04/2008
3553 3571 2846 3497 3494 3404
Tippecanoe
40° 25.0920 N/86° 53.9590 W
09/29/2009
3615
Hamilton
39° 08.6830 N/84° 30.4830 W
11/02/2009
3563
fractions, corresponding to each of the major peaks, were each evaporated to
